Transcriptional Regulation of the L-ara

• Distinguish between positive and negative transcriptional regulation • Make predictions based on hypotheses • Infer function from phenotype Pages 519-525 of textbook

Originally written by Lazar Dimitrov Example of catabolite control

1.Grow cells in minimal media with lactose as the carbon course 2. Add Glucose 3. Measure Beta-galactosidase-LacZ -Cell default setting is to use glucose for energy production Sugar Utilized

lactose galactose glycerol maltose

sorbose Regulation of cAMP Production PEP (phospho enol pyruvate)-dependent sugar phosphotransferase system-transports glucose into the cells -PTS IIAglc exists in two form +/- phosphate

-phospho IIAglc activates adenylate cyclase

-ration of IIAglc to IIAglc-P depends on glucose availability

-Hpr Histidine protein adds phosphates

Glucose

Glycolysis

PEP:Pyruvate

TCA The arabinose regulon Figure 12.18 Mutant Analysis

Galactose & Glycolysi Lactose lacY, Glucose s lacZ Negative Regulation (lacI)

L-arabinose Utilization

L- L-ribulose- D-xylulose- L-ribulose 5- 5- arabinose phosphate phosphate

Is it subject to positive or negative regulation? How Many ?

Replica plate

Minimal Mutagenesi Minimal L- s Glucose arabinose Ara-

• Test each Ara- for /recessivity to w.t. • Set up complementation tests between all possible recessive Ara- mutant pairs How Many Genes? • At least 4 complementation groups/genes (araA, araB, araC and araD) are defined by the Ara- mutants

L- araA araB L-ribulose- araD D-xylulose- L-ribulose 5- 5- arabinose phosphate phosphate

•What about araC araC- Mutants Are “Super- suppressed”

OFF OFF

C- BAD C- BAD

Plus L- No L-arabinose arabinose • What could be the function of araC? (Hint: araC is not the L-ara permease) Is araC a Positive or Negative Regulator? OFF OFF C- BAD C- BAD

No L- Plus L-arabinose arabinose ON ON I- ZYA I- ZYA

Plus No Lactose Lactose Is araC a Positive or Negative Regulator?

• What in the cause “super- suppressed” phenotype? Negative Regulation Wild type Wild Type No Plus inducer inducer

OFF RNAPol ON Y Promoter Gene Y

Constitutive Mutants Loss-of-function (LOF) mutations

RNAPol ON Promoter Gene Y Negative Regulation & Constitutive Mutants

Promoter Gene Y

No inducer - Gene will be ON/OFF? Negative Regulation & Constitutive Mutants

Promoter Gene Y

Plus inducer - Gene will be ON/OFF? Negative Regulation & Constitutive Mutants

• Partial diploid containing a constitutive and a wild type allele has wild type phenotype

• THEREFORE, constitutive mutants are dominant/recessive to wild type under negative regulation Positive Regulation Wild type Wild Type No Plus inducer inducer RNAPol

OFF ON Promoter Gene Y Promoter Gene Y

Constitutive Mutants

RNAPol Gain-of-function (GOF) mutations ON Promoter Gene Y Positive Regulation & Constitutive Mutants

Promoter Gene Y

No inducer - Gene will be ON/OFF? Positive Regulation & Constitutive Mutants

Promoter Gene Y

Plus inducer - Gene will be ON/OFF? Positive Regulation & Constitutive Mutants

• Partial diploid containing a constitutive allele and a wild type allele has a constitutive phenotype

• THEREFORE, constitutive mutants are dominant/recessive to wild type under positive regulation Hypothesis • araC is an activator of the L-ara operon

araC- - araC Plus L-ara No L-ara

OFF OFF Promoter Gene Y Promoter Gene Y Predictions •araCc will be dominant/recessive to wild type araC •Constitutive mutants, i.e. araCc, will be rare/common? •araC- mutants cannot be induced with L-ara Bahavior of araCc mutants

C+ BA D C+ BA D F’ F’ OFF ON Cc BAD Cc BAD

Plus L- No L-arabinose arabinose

Conclusion: araCc mutations are dominant/recessive to wild type araC • If araCc mutations are recessive to wild type, is araC a repressor? • If araC is a repressor, why are araCc mutations supersuppressive and not constitutive? • If araC is a repressor, why are araCc mutations rare? AraC a repressor & an activator • Revised Hypothesis: AraC is a repressor in the absence of L-ara but an activator in the presence of L-ara

Wild type Wild Type No L-ara Plus L-ara

RNAPol

AraC OFF AraC ON Promoter Gene Y Promoter Gene Y AraC a repressor & an activator

• BUT … why is araCc recessive to wild type? C+ BA D AraCc ’ F AraC AraC AraCc

AraCc OFF AraCc AraC

AraCc Cc BAD AraC

AraC No L- arabinose AraC a repressor & an activator • At least two models (not mutually exclusive) – AraC in its repressor conformation binds to DNA with higher affinity than AraCc mutants – AraC peptides form a complex (e.g. a homodimer) to be functional

RNAPol

AraC AraC OFF AraC AraC ON Promoter Gene Y Promoter Gene Y Fig 12.20